JPH02462A - Method for improving translation of mrna and utilization of the same method for production platelet factor 4 - Google Patents

Abstract

PURPOSE:To obtain human platelet factor 4 in improved expression and recovery of recombinant protein by selecting a DNA sequence having a specific length as the 1st DNA sequence of a hybrid gene. CONSTITUTION:The objective process for improving the expression of a recombinant protein includes (1) a process for constructing a hybrid gene composed of the 1st DNA sequence coding amino terminal and separated with a cleavage site sequence and the 2nd DNA sequence coding the recombinant protein, (2) a process for culturing a host cell transformed with a vector having said hybrid gene and (3) a process for recovering fused protein from the transformed cell. The above process is carried out by the following manner. A recombinant protein is coded as the 1st DNA sequence of the hybrid gene. A DNA sequence having sufficient length to inhibit the formation of undesirable secondary structure from an mRNA region and an mRNA region containing a ribosome-linking site is selected to prevent the hindrance of the approach of ribosome to said ribosome-linking site.

Description

Detailed Description of the Invention (Field of Industrial Application) The present invention belongs to the field of genetic engineering, and relates to a method for improving the expression and recovery of recombinant proteins with poor expression efficiency (expression efficiency), and a method for improving the expression and recovery of recombinant proteins with poor expression efficiency. Cloning and expressing 2,
Furthermore, the present invention relates to plasmid vectors for production using microorganisms.

BACKGROUND OF THE INVENTION Platelets are circulating blood cells that function in response to injury to blood vessels in the normal blood coagulation mechanism.

Platelet alpha granules contain platelet 4 (PF-4 or PF4), platelet-derived growth factor (PDGF), binding & fl tissue-activating peptide (C-1), and β-t [
secretes many important proteins, including ff-boglobulin (β-TG). The biological roles of these proteins (one of which has substantial amino acid sequence homology) are not fully clear, but their four most important properties are as follows: is: (a) os
a by stimulating synthesis and cell division.

ability to act as a potent mitogen, (b) Gulni]
-stimulates a variety of metabolic activities in tether m cells, including gas transport, glycolysis, production of prostaglandin E2 and cyclic AMP, synthesis of hyaluronic acid and glycosaminoglycans, and production of plasminogen activator. (C) by its ability to attract immune system cells to the site of inflammation and stimulate their function, and (d) by its high affinity for sulfated glyco-1 saminoglycans, an important component of the cell surface.

Ability to bind to connective tissue 11 and mast cells.

Platelet factor 4 acts as a procoagulator.

It is a platelet secreting kumbak that has several biological functions such as antiheparin action, immunoregulatory action, and chemotaxis. P
Regarding F-4, the following is proven:
It is a potent chemotactic agent for mononuclear cells, L and neutrophils (Duel et al. (1981) r'roc NatlQ
cad Sci USA 78: 4584):
Binding to serotonin (IIemsLra, V
, L. (1983) Thrombosis Re529
: 323); Reconstructing two I lagenases [
1110-11arper, J. et al. (1978) 3ci
e-li-131st! 'j:991); reversing immunosuppression in animals (Katz, 1.R, et al.
1986) 1'roc Natl Acad Sci
tl! Q--β3:3491); and inhibiting the growth of certain lung tumors (Taylor, S. and F.
olkman, J. (1982) N-a+,u
re 297: 307), PI'41, isolated from rami blood and human blood and purified to near purity (Wu, V, Y, et al. (1977), Pre.
pBjochem-re 479; and 1evine,
S, P, and -〇old, ■.

(197G), J. Riol Che1251:
324), and its amino acid sequence and physicochemical properties were determined [e.g.
(1977), Throw Res 11:89
Please refer to 3]. Recently, the DNA sequence of a cDNA derived from a human erythroleukemia cell line was determined by R'oncz, M. et al. (1987), β1ood 69: 21.
9), was announced. This sequence has a leader sequence encoding 30 amino acids preceded by an initiating methionine residue, followed by a coding region encoding a protein of <70 amino acids.

To date, there has been no report of successful production of PF-4 by recombinant DNA technology. Furthermore, unlike the success achieved with human connective tissue activating peptide-1it, a protein with >60% homology to PF-4, the present inventors using PF-4 in the same expression system Similar attempts at direct expression of PF-4 were completely unsuccessful. Although PF-4 reported in early studies was obtained from platelets, the isolation method is not practical for producing large quantities of the protein. In this regard, it is an object of the present invention to provide a means for producing PF4 by recombinant DNA technology. This means that the synthetic gene encoding PF-4 (P
F-4); and PF-4 fused to the modified colicin El structural gene region.
This includes the use of an expression vector containing 4 genes.

The plasmid derived from recombinant pBR322 is pBI13
22 and a DNA fragment ↓ fragment 1 containing the colicin E1 expression control sequence and colicin structural gene inserted into pBR322 at the Pstl site of pBR322, Waleh
, N.S., and Johnson, P.H.

(1985), Proc Natl Acad.
Sci IJSA 82: 8389-
8393 and International Publication WO851010
67. This plasmid was used to express a synthetic gene encoding human connective tissue activation peptide-■.

U.S. Pat. No. 4,366,246 describes a recombinant method and method for producing by a microorganism a fusion protein of a foreign polypeptide linked to a foreign amino acid sequence through amino acids that define specific cleavage positions. Generally described.

A number of references have been cited by Brosius J, by which a desired gene can be combined with a portion of another gene (e.g. 1acZ, MS2 polymerase, phantom).

The situation is supported that a more stable hybrid protein is produced by fusing it to mE or the lambda cl repressor). In the above literature.

”IExpression Vectors Empl
oying Lambda-, υfurther.

Iac-, and b-Italy-Derived Promo
ters,” Rodriguez.

R, L, and Denhardt, D, T, 1i;
and Vectors: EightSurveofMole
cularC1oninVectorsandThei
rUses, Boston: Butterwor
ths; 1988; 207.

This reference does not address the low yield of product resulting from inefficient translation of Mensenger RNA.

(Structures of the Invention) One aspect of the present invention is a method of improving expression of 5&11 recombinant proteins. This method generates a hybrid gene consisting of a first DNA sequence encoding the amino terminus and separated by a cleavage site sequence and a second DNA sequence encoding the recombinant protein (the hybrid gene coats the fusion protein). A method comprising the steps of constructing a vector containing the hybrid gene, culturing a host cell transformed with a vector having the hybrid gene, and recovering a fusion protein from the transformed cell. 1, the mRNA 1U region encoding the recombinant protein and the mRNA region containing the ribosome binding site inhibit the formation of an unfavorable secondary structure that would prevent ribosomes from accessing the ribosome binding site. selecting a DNA sequence of sufficient length.

Another aspect of the invention is a plasmid expression vector.

This plasmid expression vector, when inserted into E. coli, expresses the product of the fusion gene in an amount sufficient to provide at least 10% of the total cellular protein.

A preferred embodiment of the above recombinant plasmid expression vector is Col(150)-PF4. This is p8R3
pBI1322; and a DNA fragment containing the colicin 1 structural gene (truncated at the carboxyl terminus) fused to the colicin El expression control sequence and the sequence encoding platelet factor 4;
including.

Another aspect of the invention is a method for microbial production of platelet factor 4, the method comprising:

a) a step of culturing E. coli transformed with the above expression vector, and b) a step of disrupting the cultured transformant.

C) Purifying the fusion protein from other cellular proteins.

d) cleaving the fusion protein at a specific cleavage site; and e) recovering platelet factor 4 from the cleaved product of step (d).

Yet another aspect of the invention is the product of the above method.

In particular, the fusion Cot: PF4 metsenger RN^ transcript and gene product, and the platelet factor 4 itself recovered therefrom.

The method of the invention involves producing PF-4 in the form of a fusion protein. The endogenous portion of this fusion protein is a biologically inactive colicin E1 fragment that contains a selective cleavage site within the fusion portion so that the desired protein is easily cleaved. Such a protein is produced by inserting the PF-4 gene with an appropriate translation couminator into a vector (containing a portion of the colicin structural gene with a convenient restriction enzyme site near the carboxy terminus and a colicin expression control sequence). can be prepared using expression vectors. Due to the high affinity of PF4 for glycosaminoglycans, especially heparins, heparin and chondroitin sulfate can be used for affinity chromatographic isolation of fusion proteins and free PF-4.

As used herein, the term "U platelet factor 4"
PF-4 refers to a protein consisting of 70 amino acids that substantially corresponds to the mature protein described by Poncz et al. means having any of the biological activities related to

Removal of amino acids inserted into a sequence during translation.

It is also possible to change the initial structure itself by addition or modification. This can be done without impairing protein activity. Such substitution or other modification may result in
A protein having an amino acid sequence that falls within the definition of "having an amino acid sequence substantially equivalent to that of rPF-4" is produced. For example, Poncz
According to the literature of et al. (supra), residue 47 was recognized to be correctly asparagine. However, this position had previously been identified in the art as aspartic acid or asparagine, a fact that was not known in the literature regarding PF-4. In the present invention, 47
We synthesized PF-4 sequences with each residue at position 1 and found that each protein has biological activity and that the protein with aspartic acid at position 147 seems to have more desirable solubility. .

As with all proteins, the PF-
The exact chemical structure of the 4 protein depends on a number of factors.

Having ionizable amino and carboxyl groups in the molecule, proteins can be obtained as acidic or basic salts or in neutral form. All such preparations that retain their activity when placed under suitable environmental conditions are
1) Included in the definition of F to 4. Furthermore, the original amino acid sequence can be modified more generally by derivatization with sugar moieties (glycosylation); derivatization with other complementary molecules such as lipids, phosphate groups, acetyl groups, etc. Its size can be increased by combining it with saccharides. The initial amino acid structure is also. May aggregate to form complexes.

Such increased size aspects are achieved through post-translational processing in the production host. Such other modifications may be introduced in vitro. Additionally, individual amino acid residues in the chain are oxidized.

Can be modified by reduction or other derivatization.

Such modifications do not result in loss of activity.

It does not exclude protein sequences from the definition.

Various attempts have been made to express PF-4 by recombinant DNA techniques, including direct expression of PF-4 (ie, not fused to other peptide sequences) in bacterial hosts. Using the same expression system used for human CTAP-III, no significant levels of PF-4 were obtained.

This is not surprising given the fact that CTAP-fl has extremely high homology to PF-4 (almost 75% identical in nucleotide sequence and 65% identical in amino acid sequence). There was.International publication WO8510
The colicin E1 expression system disclosed in 1067 was found to produce approximately 30% of the total cellular protein CTAP-I. using sensitive radiolabeling techniques and in vitro transcription-translation assays.

The low yield of PF-4 protein is a result of poor or inefficient translation of the PF-4 messenger RNA (a+RNA) and is not due to poor transcription efficiency or protein instability. It became clear.

Studies have shown that gene expression is often enhanced when the secondary structure of the mRNA exposes translation start-determining sites, but reduced expression when these regions are masked. Zucker, M., and SLi
egler, P. (1981) Nuc Ac1d
By the method of Res 9:133, using a computer method to predict the secondary structure of single-stranded nucleic acids
The structures of F-4 and CTAP1116Dn+RNA were predicted. The results are shown in Figure 1 (CTAP-m) and Figure 2.
The ribosome binding site of CTAP-m mRNA is predicted to be exposed in the form of a single-stranded loop, but the ribosome binding site of PF-4 mRNA is shown in a distant sequence (nucleotides 232 to 237). .Figure 2)
It is predicted that ribosome binding is inaccessible by forming base pairs with.

-i, alteration of the mRNA sequence and/or its secondary structure near the ribosome binding site and the AUG initiation codon is expected to affect 1 expression levels.

Therefore, when 1 expression levels are low, one skilled in the art will generally recommend m
Attempts to modify the nucleotide sequence of RNA. Until now, it has not been recognized that "golden" interactions, ie, long-range interactions between the above-mentioned ribosome binding sites and mRNA regions outside the local structural region, affect translation efficiency. The present invention: overcomes the problem of poor translation of mRNA by: In other words.

Unfavorable interactions that fold RNA can result in the insertion of a DNA sequence of sufficient length between the DNA containing the ribosome binding site and the DNA encoding the poorly expressed gene, creating a hybrid gene encoding the fusion protein. This causes an interruption. As used herein, "sufficient length" indicates that the inserted DNA sequence must be larger than 15 base pairs to separate the translation initiation codon and the beginning of the recombinant protein coding sequence. . Preferably, this distance is within a range of about 150 base pairs to about 450 base pairs.

An appropriate expression vector is used to construct the fusion gene such that the fusion gene is flanked by transcription initiation control sequences at the 5° end and transcription and translation termination sequences at the 3" end. (5゛- and 3゛- intend the direction of transcription).

(Blank below) After preparing the plasmid DNA for introduction into a suitable host, the host was transformed and cloned.

The clones are cultivated and the desired product 1, e.g. l'F-
Individual clones showing effective expression are selected by detecting the production of 4. By way of example for the present invention, effective production means that the expression level of the PF-4 fusion protein is at least 10% of total cellular protein. Screening can be effectively performed using Western blotting (antibody detection of product) of host cell colonies transferred to filters of nitrocellulose or other suitable material. Alternatively, analyze samples using gel electrophoresis to quickly determine which clones are most efficiently expressed by visualizing protein staining intensity, autoradiography, or Western blotting of product bands. can be compared directly. Although this screening procedure is usually sufficient, more quantitative immunoassays or enzymatic assays can be used if appropriate.

In preferred embodiment B of the present invention, the insertion 11N^ is
PF-,1 and truncated at the carboxyl terminus] encodes the ricin E1 protein. In these embodiments, the corytan El gene encodes a protein that has a hydrophobic (a physicochemical property that facilitates purification of the fusion protein from other cellular components) portion (approximately 30 amino acids). This hydrophobic moiety allows selective extraction of contaminating proteins from the fusion protein using high salt concentrations (e.g., 1 M guanidine hydrochloride) and yields colicin EI PF-4 with a purity of over 80%! ! l! Combined protein is provided.

After partially purifying the fusion protein, PF-4 is isolated by subjecting the fusion protein to a chemical or enzymatic reaction that cleaves the protein at the fusion site. For example, if one fraction of the protein is fused by a methionine residue, the protein can be treated with cyanogen bromide.

Below, PF-4, Coritan El fragment and PF-4
and P produced by microorganisms.
A detailed description of embodiments of the invention involving the purification of F-4 is provided.

The synthetic gene for Idanzi ψ-construction PP-4 consists of two major subfragments (I
It consists of a 243 base pair DNA constructed from The 12 oligonucleotides were synthesized using the phosphoamidite method from Applied Hiosystems D
Synthesized with IIA Synthesizer.

Twelve synthetic oligonucleotides were purified and ligated to prepare fragments 1 and 2, as described below and illustrated in FIG.

Oligonucleotides F and 1, purified 7p+rl, 90mM with 2mM EDTA!
- A polyacrylamide gel (12%) was prepared using lysoborate buffer. 1 to 5^2O medium crude Olivoscleoside sample and the same amount of 7M urea were added to 10
Mix in mM Tris-+1 CIil solution (pu 7.5). Add DNA X14 to the gel and add 1 dye mixture (0
, ] 77% Bromophenol Fluor 0.27%
7,5) was added to one of the wells and the rate of migration of the oligonucleotide was monitored. 400 ~ until the bromophenol blue moves approximately 30 cm from the tip of the gel.
Electrophoresis was performed at 600v.

The gel was removed from the plate, wrapped in plastic wrap, and plated onto a fluorescent bank.

DNA was visualized using short wavelength ultraviolet light. The desired hand was carefully cut out with a razor blade. This gel piece was placed in an Enbendorf tube and crushed with a glass tip. Then 0.5 ml of TE (10mM) cis11
CI, 1mM EDTA, pH 7.5) was added to this sample.
In addition to bu.

The tube was rotated overnight to extract the DNA. The tube was centrifuged for 10 minutes at 15,000 rpm and the supernatant was collected. This DNA sample is ip
After diluting the mixture 10-fold with C-185ep-Pak column, it was desalted using a C-185ep-Pak column. DNA recovery rate is generally 50%
and 80%. The eluate was lyophilized and then resuspended in 0.5 ml of water.

The purity of the recovered DNA was confirmed by autoradiography after gel electrophoresis of the 32p-phosphate labeled product at the 5' end by T4 kinase.

2. Oligonucleotide ゛′・t The reaction mixture for linking the oligonucleotide is 5
0d Tris-)ICI, (p)l 7.5), 10
mM MgCb.

20mM dithiothreitol, In+M ATP,
Consisting of 100 picomoles of DNA (5° end concentration) and 100 units of T4 ligase, the total volume is 10
0μ! Met. These reaction mixtures were incubated overnight at 16-21°C.

The ligation reaction was terminated by adding a 3-fold excess of EDTA to chelate the +'g''. One sample was heat denatured after addition of the urea and dye mixture and analyzed by gel electrophoresis.

Figure 3 shows the 12 oligonucleotides used to construct the 9 synthetic genes. Oligonucleotides 1-8
constituted fragment I, and oligonucleotides 9 to 12 constituted fragment II. These two fragments were constructed in vitro and then cloned into the 1M13 vector as described below, and the DNA sequences were confirmed.

A double-stranded replicative form (RF) DNA of the old cloning beta ML3 was prepared as follows. E. coli J with M13 introduced
MIOI cells were inoculated into 2xYT broth and cultured overnight at 37°C.

Cells were harvested by centrifugation, washed, resuspended in buffer, lysed with lysozyme and Triton X-100, and treated with ribonuclease. remove cell debris,
RF DNA was purified using +CsCl-ethidium bromide equilibrium centrifugation. Ethidium bromide was removed by extraction with n-butanol. I? F
The DNA was dialyzed and concentrated by ethanol precipitation.

E. coli was cultured in 2xYT pros until 0D66° was between 0.6 and 0.7. Cells were harvested by centrifugation, resuspended in 50mM CaClz (half volume of medium), and kept on ice for 20 minutes. Cells were harvested and resuspended in 1/10 volume of CaCl□.

Dish pi transduction pre-endonuclease Eco Rr and X ha
M13 RF DNA digested with PF-4
ligated to fragment I or ■ and competent JMI
Mixed with OI cells and kept on ice for 20-40 minutes. This mixture was heat dried at 46°C for 2 minutes.
PTG, Blue-gal.

Soft agar (46°C) and freshly grown JMIOI
mixed with cells. This mixture was plated on YT agar plates and incubated overnight at 37''C.

JMIO transformed with M13 without PF4 fragment
I cells synthesized β-galactosidase and gave blue plaques. Cells transformed with old 3 containing the PF-4 fragment did not produce β-galactosidase and gave colorless plaques.

Nitrocellulose filter hybridization (margin below) Recombinant phages were screened for the presence of the PF-4 gene sequence as follows. Phage cultures are
BRL (Bethesda Research Lab
The dots were plotted onto nitrocellulose filter paper using a 96-well HYBRI-DOT manifold from the Oratories. The plotted phages were lysed and the DNA was denatured and immobilized by washing the filters once each with stirring for 15 min in the following solutions: 0.5 M NaOH;
0.5 M Tris, pH 7.4; 2X5SC
(0.3M NaCl, 30mM sodium citrate), pH 7. These filters were briefly washed in 95% ethanol, air dried, prehybridized for 3 hours, and incubated with 32p-labeled oligonucleotides overnight at room temperature. Hybridized with. These hybridized filters were treated with 0.1% SO
5 for 15 min at 25°C in an I
Washed twice, dried, and autoradiographed. The filters were then washed again and treated with the oligonucleotide 5'-GTAAAATCTGTCTA.
This was investigated using GACCTG-3' as a probe.

This oligonucleotide.

Corresponds to the junction between the PF~4 (■) and PF~4 (U) subfragments.

! 1:01 M
9 medium (per le: NLCI 1 gt NaJP
IL'LO6g.

43 g of KHzPO, 5 g of NaCl, 3 g of casamino acids, 10% Mg5On ld, plus 20% glucose io- and lM CaCIz after autoclaving.
in an incubator at 537°C at
The cells were cultured until the cell density reached approximately 5 XIO@CFtl/d. Chloramphenicol.

It was added to a final concentration of 100 ug/1 afl, and the culture was continued at 37°C for an additional 6 hours. 5har
Cells were collected using a PEL continuous centrifuge. 10
g (4 wet weight) of pellets, 50n+M EDTA
180sj including 15% Siyotang! 5On+M
) was suspended in Lis-○CI buffer (pH 8,0).

Next, 0.14 g of lysozyme was added.

The mixture was allowed to stand at room temperature for 10 minutes. Then 10%5
OS 16d and 5M potassium acetate 20- were added. After incubating this mixture on ice for 30 minutes, the 5S-
Centrifugation was performed for 30 minutes at 12 OOOrpm using a 140-meter centrifuge and a 5-orbit centrifuge. Add 4 μg of pancreatic ribonuclease to the supernatant and heat the mixture at 37°C.
I stayed indoors for an hour. 0.1M1-lith (p
This sample was extracted twice with the same amount of phenol saturated with 3.1 + 8.0 ) and 1710 volumes of the sample were extracted twice.
The DNA was precipitated by adding 0 M sodium acetate and 2.5 volumes of cold ethanol, followed by -20
'C overnight. 7.00O by cooling 5orva l l centrifuge using HB-40
The precipitate obtained by centrifugation at rpm for 50 minutes was collected. This pellet.

10m containing 0.3M NaCl and 5mM ED T^
M't-Squirrel oc+ 11 street solution (pH 7,5) (
NEII solution) 50d. This sample was then transferred to a 5M 100cm Bio
-Get A, 5 columns (Bio-RadLabor
Atories, Richmond, CΔ). DNA was eluted with NE buffer. Both volumes of 20 ml were collected at a flow rate of 60 d/hr. Elution of DNA is
Each portion was monitored by measuring the absorbance at 26On+m using a G11ford 2600 UVVis spectrophotometer. Both host DNA and plasmid DNA were recovered in the void volume. Both portions containing DNA were pooled and precipitated with ethanol. 8. In a refrigerated 5-orva l centrifuge using an HB-40-taper.
Collect the precipitate by centrifugation at 00 rpm for 40 minutes,
5-10mM Tris-11 containing 0.2M NaCl
Redissolved in 01 buffer (pH 7,8).

This DNA sample was transferred to a 0.9M90cm RPC-5
After applying to the column, it was packed at 30"C under pressure. DN
A is 10IIIFI Tris-11cI buffer (P
Elution was with a linear gradient of 0.6-0.7M NaCl in H7,8) (total volume 1ffi). Both portions of 2.5 d were collected at a flow rate of 0.8 d/min.

DNA elution was performed using a conductivity meter (Radiometer,
Copenhagen) and by agarose gel electrophoresis using a vertical agarose slab gel (0.25 x 14 x 15.5 cm).

These samples were mixed with lITIM EDTA and 5mM
40mM Tris base buffer fL containing sodium acetate
(pH 8,2) (Put it on 1% agarose prepared in TAE llll liquid and apply it at a constant voltage of 5 V/cm for 3 hrs.
Time electrophoresis was performed. Both fractions containing supercoiled ON^ and nicked circular DNA were pooled separately and precipitated with cold ethanol. The resulting precipitates of Cot EI DNA molecules were diluted to 1, 0 ml and 0 ml, respectively.
, 6 tnl of TEN buffer.

E.coli strain 294 (pBR3
Plasmid pBR322 was isolated from 22).

200uf of Col EI DNA!
Circular Col El -DNA with a slit (0
, 7 gg/ul) and 2.8 d of 0.3 M sodium acetate were mixed. This oral MA solution was mixed with an Omnimixer (DuponL Instruments, New
The DNA was then sheared at 38.50 rpm for 20 minutes.

The temperature was maintained at 0°C throughout the shearing process. The sheared DNA was precipitated with ethanol and 100 μi TEN
Redissolved in buffer and added calf intestinal phosphatase (CIT) (Boehringer Mannhe
i+ll, Indianapolis, IN). Treatment with CIT consists of two reaction mixtures 500
μ! It was carried out inside. Each reaction mixture was mixed with 380 μA of distilled water.
, 1M Tris-+1cI buffer (pH 8,0) 50μ
2. Zinc sulfate 10mM 5ul, CIT (IO
U/μj2) contained 5ul. After incubation at 37°C for 30 min, an additional 5 μi of CIT was added.
Incubation was continued for an additional 300 minutes at 37°C. These reaction mixtures were extracted twice with phenol saturated with the same volume of buffer, and the DNA was precipitated with ethanol. D.N.
A heterogeneous population of A fragments.

Further purification was performed by sucrose density gradient fractionation.

Separated according to size. A discontinuous CJ sugar density gradient was placed in a centrifuge tube for a 5W40 rotor (Beckman).

It was prepared by sequentially layering 3.4 d of 20%, 15%, 10%, and 5% sucrose into 0.3 M sodium acetate buffer (pH 1.0) containing 1 mM EDTA. 100 lE (D of 0.25 μg/μ
NA sample.

Layered on this sucrose density gradient, L 8-70 B
20 min at 10°C using an Eckman ultracentrifuge.
Centrifugation was performed at 35.00 rpm. Both 0.5 ml portions were collected and precipitated with ethanol. These precipitates were redissolved in 50 μl of TEN buffer and analyzed by agarose gel electrophoresis. Bacteriophage D
A DNA fragment obtained by treating NA with μnd ■ endonuclease was used as a bimolecular weight standard. The λ/Hindll1 reaction mixture contained 27μ2 of distilled water,
5 x stone dll! 10μl of buffer solution, λDNA (0.7g
g/μl) 1μ! , and l1indnl solution (2U/
μ12μjl! Contained.

Contains on average 2.000 bp sheared ColEI DNA fragments.

Sucrose density gradient fractions were pooled, ethanol precipitated, and redissolved in TEN buffer.

Cloning of ColE1 into BR322 Plasmid ρBR322 was cut with PstI into a linear molecule. The reaction mixture contained 520 μj of distilled water! , 5X
200 μl of Pst) buffer, 200 pf of pBR322DN Hachihe solution (0.25 μg/μ1), and Pst I
(12U/aE) containing 80μl, 37
Incubated for 4 hours at °C. 20m of EDTA
The reaction was stopped by adding up to M and extracted with the same amount of phenol. Precipitate the DNA with ethanol.

Redissolved in TEN buffer.

Distilled water 5μE, 500mM potassium cacodylate 20
μ21OIIIM cobalt chloride 10μl, in+M
DTT 10μ41! , 10mM dGTP2μ
f, GoIt-dGTP (New England
nd Nuclear Corp.

ration) 20ul, DNA (0,04
Mg/ul) 25u 1 and terminal deoxynucleotidyl transferase (Bethsda R
e5earch Laboratories, Inc.
,.

Gaithersburg, MO) (12U/
u l ) linear p in the reaction mixture containing 5 u l
Poly(dG) homopolymer was added to the BR322 molecule.

PoIJ (dC) was added to ColE1 sheared fragments in a similar reaction mixture except that the total DNA was 2.0 μg and the nucleotide triphosphate was dCTP
Added homopolymer. The above reactions were carried out at 37°C.

2 minutes and 3 minutes, respectively, and 20 m of EDTA.
Stopped by adding up to M and extracted with phenol. ColB1- (poly(dC)) fragment,
Redissolve in 115μf of distilled water and add 0.5M NaCl.
40μm, 50mM EDTA (pH 7,25) 4
0μ! , and straight pBR322-[poly(dG)
) DNA solution (0,1μg/μf) 3μ! By adding linear pBR322-(poly(dG))
Annealed to the molecule. The annealing mixture was incubated at 70'C for 15 minutes and then cooled to 40'C over 5 hours. The mixture was kept at 45°C overnight and then cooled to room temperature.

For transformation into E. coli 294, cultures grown overnight in L-broth were diluted 1:100 into fresh pure single-gloss medium at an OD of 6°. is 0.6
The cells were cultured at 37°C without shaking until . At this point, culture 35Id was incubated at 6.0°C for 120 min at 4°C.
Centrifuge at 0 Orpm and add the pellet to 0.05M Ca.
Resuspend in 20ml of C1g. After incubating these cells on ice for 15 min, 4.000 rp
The samples were collected by centrifugation for 10 minutes at m. These cells were resuspended in 4 ml of 0.05M CaCl, 50μ2 of annealing mixture, and 10mM MgC.
1hM) Lys-1 containing h and 10mM CaCIz
1cI (pH 7,5) and 200 μ2 of a DNA solution prepared by adding 150 μ2.

The mixture was incubated at 0°C for 25 minutes, then at 50°C for 10 seconds and at room temperature for 10 minutes. At this point, L-broth 14- was added and the culture was shaken for 30 minutes at 37°C. Next, 1
．． 25mg/ml Tetoshu Iclin) Volume: 480mg/night
II E @, 1 J■ was added to this 111 feed and inkcutting was continued for an additional 30 minutes. 100/! A JII amount of A was plated on freshly prepared agar plates containing 25 ml of L-broth, 1.1°596 agar, and 25 t g/d tetracyclinol. Furthermore, 25μ
The sensitivity (Aρ'') of the TeI racycline transgenic (Te') transformants to ampicillin was determined by plating on agar-F containing ampicillin of ε/R1.

Tc'Ap' transformant colonies were then screened for spontaneous production of colicin. Single colocii were spotted onto L-agar plates and incubated overnight at 37°C. These colonies were killed by exposure to chloroformus and then plated on 0.796 agar r:E,c.
Oli K-12, CL142 overnight culture was overlaid with 0.1-ml of broth containing 5-ml of broth. After the agar had hardened, the plates were placed in an incubator at 37°C overnight. Colonies with an inhibition circle around them are called colicin producers (
Cot”).

By analyzing a small amount of clear trisate by agarose gel electrophoresis, Tc'Ap'Col' transformed colonies were identified. 411! ! ! Screened for the presence of Eblasmi F. What is the size of the plasmid?

Eight plasmid standards (Mar
einaF, [,, et al. (1978), P! asmid
Upper: 411-420) was used to determine the mobility of DNA in an agarose gel by electrophoresis.

Transformed clones were grown in 2p medium.

1j artlysate was prepared as described above.

Add the supernatant to pancreatic 5IRNase A (100u fX/
DNA was precipitated with ethanol and extracted with phenol.
EN ill liquid-liquid redissolved.

Restriction enzymes are from Bethesda Re5search
Laboratories.

Obtained as a commercial preparation from Ir1c, (BR,c). Using the conditions specified by BRL2, this D
NA, Pstl, EcoI? I, Smq
Digested with I and Sac 17.

Samples were run on a 1% agarose gel and subjected to electrophoresis at a constant voltage of 5 V/cm for 4 hours. The molecular weight of the restriction fragment is
Determined by comparison to standard migration patterns of bacteriophage λ DNA digested with Hindll and Hae III.

(2) Restriction analysis using Pstl was employed to establish the size of the insert into the fly 1 site of the pBR322 portion of the recombinant plasmid. Figure 4 shows one of the transformed clones.
'It is. Restriction enzyme map of the recombinant plasmid named pNP6. It was named NP6-294. This transformed t:z −:/ sample was 198
It was deposited with the ATCC on August 24, 2003. This sample was assigned ATCC accession number 39418. This deposit is accepted under the Budapest Treaty, will be maintained in accordance with its provisions, and may be transferred to third parties.

E. coli NP6-294 strain (pNP6) was grown, and 500 ug of DNA was added to TEN 11 buffer 3.9.
d, 3.45 g of CsCl and Echijiu J,
Plasmid DNA was further purified by adding 0.1 mR of Promid stock solution (5 ml/ml). This mixture was poured into a 6-port acid cellulose tube for 5W50.1tV-ta (Beekman) and centrifuged at 36,000 rpm for 40 hours at 10'C. Plasmid DN
A's hand was placed under a long wavelength ultraviolet lamp, and the tube was removed with a syringe by applying 711+ from the side. This DNA sample was extracted five times with butanol,
Dialysis was performed against 100 volumes (x3) of TEN buffer for 24 hours at 4°C. INNΔ was then precipitated with 2.5 volumes of ethanol and 1/10 volume of 3M sodium acetate.

The P and W-attached 1N sub-A-R=IO preparation plasmid pNP6 contains two EcoR ■ restriction sites, one of which is colicin EIX! one located in the carboxyl terminal region of the i gene, and the other located near the tetracycline resistance gene of the original pBR322 vector.

A derivative of pNP6 lacking the second site was constructed as follows. pNI under restriction reaction conditions to obtain a linear molecule (cleaved at only one of the two sites).
'6 Ecol? Digested with l. The linear molecule of pNP6 was purified by agarose gel electrophoresis,
Single stranded ends were filled in by reaction with NA polymerase 1 and deoxyribonucleotide triphosphate. The resulting molecule was cyclized in a blunt end ligation reaction using T4 ligase and then transformed into E. coli 294 as described above.
was used for transformation.

Colicin-producing transformants were selected as described above.

Isolate DNA from individual clones and insert a single unprocessed Eco into the colicin gene by digesting with EcoRI.
It was identified that the DNA contained the R1 site. single E
The location of the coR1 site was further confirmed by restriction endonuclease mapping.

The synthetic PF-4 gene was removed from the old 3-sequencing vector of NF2-Col 504-PF 4 and cloned into the single EcoR site of plasmid pNP6ΔR1. pNP6- Col (504)P shown in FIG.
It was named F4. This recombinant DNA plasmid is
PF-4 protein (70 amino acids) and colicin residue 1
It has the ability to produce a large protein (574 amino acids) fused with ~504. Cells containing this plasmid were grown in culture and treated with mitomycin C to induce synthesis of the fusion protein.

Small-scale experiment (Fig. 5, lanes 2 and 3H)
The fusion protein is produced at a rate of about 10-20% of the total cell mass. However, the purification and characterization of the CNBr cleavage product from the PF-4 fusion protein by phosphoaffinity chromatography could not be reproduced due to the formation of erroneous disulfide bonds and the interference of colicin fragments. The lower the plasmid, the higher the medium capacity (e.g., because the construction of this plasmid may be unstable).

One dose was also related to the use of 121).

NF2-Col (150) -PF 4 i! The yield of IM correctly folded active PF-4 protein is
The improvement was mainly achieved by reducing the size of colicin segments. Under partial EcoRV digestion conditions approximately 1
By deleting ,000 base pairs of DNA, p
An intermediate size fusion gene was constructed from NP6 Col (504) PF 4. pNP6-Co
There are three EcoRV restriction sites in (504)PF-4; two within the corytan E1 structural gene and one within the tetracycline gene, as shown in Figure 4. do. Since transformants are selected by Tc', only desired transformants in which the EcoRV fragment has been deleted from the colicin gene are obtained. The resulting plasmid is named Col(150)-PF4.

It encodes only the 150 amino acids of colicin that precede the PF-4 code sequence, and the colicin segment and PF
Contains a single methionine residue associated with -4. Fusing this colicin segment to PF-4 Kumbaku particularly improves recovery rates and purification procedures.

Plasmid pHP6-Col (150)-PF
iE, Coconut 294 cells containing -4 were reproducibly cultured (in a volume of up to at least 121 cells) and upon induction with mitomycin C.

Col(150)P accounts for approximately 20% of total cellular protein.
F 4 fa compound was produced.

(Left below) NF2-Col (57-PF4 i, produced plasmid pN
P6 Col (150) 5s in PF 4
By deleting the region between tn and the EcoRV restriction site, the size of the colicin fusion peptide was further reduced by 5
It was reduced to a segment consisting of 7 amino acids. Purified pNP6-Col(150) PF4 loNA,
5stI [cleaved into a linear molecule with restriction enzyme 5 and then T
4 DNA polymerase and dCTP. Under this treatment condition, the 5sLII-generated ends of the plasmid DNA were converted to blunt ends by the 3°-5' exonuclease activity of the polymerase. The product of this reaction DN
A is digested with EcoRV restriction enzyme, the obtained large NA fragment is purified, and a circular D is obtained by DNA ligase reaction.
Convert to NA and then pNP6 Cot(150)
PF4 was used to transform cells as described.

The resulting plasmid was pNP6-Cal (57)
Referred to as PF4, it encodes the 57 amino acids of colicin that precede the PF-4 cod sequence and contains a single methionine residue that connects the colicin segment and PF-4.

pNP6-Cot (150)
PF 4 to 1M guanidine hydrochloride (GnHCl) or 2511M Tris.

10mM EDTA, 50mM glucose, pH
The induced cells were prepared by sonication in 8,0 (TεG).

The fusion protein was found in the insoluble fraction. After solubilizing the PF-4 fusion protein in 7M urea or 6M GnHCl, the protein sample was extensively dialyzed against distilled water for the CNBr cleavage reaction. The fusion protein precipitated during dialysis was collected by centrifugation. This precipitate was lyophilized, dissolved in 70% formic acid, and reacted with a 100-t, ooo-fold molar excess of CNBr at room temperature for 18 hours.

The CNBr reaction mixture was lyophilized and resuspended in distilled water;
It was then freeze-dried again. This protein mixture, 6
0.05 M Tris buffer (pH
8,2). A reducing agent (e.g., dithiothreitol or β-mercaptoethanol) is added to a final concentration of approximately 0.1 M, and the mixture is incubated at 20°C and 37°C.
The cells were incubated for 1 to 4 hours at a temperature between .

The resulting solution was then dialyzed against the same buffer without reducing agent. This tannori/8 liquid is PF
-49ff degree is about 0.2 mg/d.

and a 2:1 mixture of oxidized and reduced glutathione was prepared to a final concentration of 1 mH. The protein folding reaction was allowed to proceed for 10-20 hours at room temperature. During this time, 7 volumes of protein solution were added to 10 to 50 volumes of 0.05 M NaCl containing 0.5 M NaCl. −Lys buffer (
Dialyzed against pH 8.2). Due to these conditions,
PF-4 with correct disulfide bond formation and folded into the original structure was obtained with a yield of 80% or more.

The heparin affinity chromagraph folding mixture was subsequently dialyzed against distilled water and lyophilized. The protein was then dissolved in GnHCI or Na(l) or chondroitin sulfate at high concentrations (>10 mg/d Tannomac) immediately prior to purification by heparin affinity chromatography.

Diluted to an ionic strength of 0.2-0.4. The chromatography column was made of commercially available heparin agarose (BioR
ad). Heparin may be substituted with other glycosaminoglycans (eg, chondroitin sulfate). Therefore, as used herein, use of the term "heparin" also includes related compounds.

Proteins were applied to a column in a low ionic strength buffer (pH 6,5) and the column was eluted with a linear gradient of NaCl. Folded transgenic PF-4 was dissolved at NaCl concentrations between 1.2M and 1.4M. This concentration is.

This position corresponds to PF-4 isolated from human platelets. Unfolded or misfolded PF-4 has significantly lower NaCl concentrations (typically 0.7
molten with NaCI of M or less.

Tiny PF-4 prepared using this procedure was analyzed by reverse-phase HPLC, amino-terminal sequence, amino acid composition, and reactivity to human PF-4 antiserum to show that human PF-4
It could not be distinguished from F-,1.

The purified rice purified PF-4 protein is typically formulated with a pharmaceutically acceptable carrier for administration to humans, eg, to stimulate wound healing. Typically, conventional topical formulations (e.g. creams).

They can be administered to the desired site in the form of pastes, gels, sprays, ointments, and skin creams). Carriers used in such formulations are known and include, but are not limited to, petrolatum, polyethylene glycol, gelatin, isopropyl myristate, polyvinyl alcohol, etc., or purified PF-4 protein. may be administered using a modified release dosage form, typically a bandage or skin patch containing PF-4 so that it is released into the skin at a controlled rate. The regulatory mechanisms are diffusion, osmosis, dissolution, and erosion.

Or it can be iontophoresis. PF, -4 topical formulations contain a small number of additives (e.g. emollients, stabilizers.

surfactants, skin permeability improvers, and pigments). The concentration of PF4 in a formulation is a function of the prescribed dose and the surface area treated. This concentration is usually within the range of 0.0001 to 1% by weight of the dosage form. In any event, this V injection is sufficient to produce the desired pharmacological effect (eg, chemotaxis, etc.) to promote wound healing.

Purified PF-4 Kumbak is also available. Ii! ¥! It may be formulated for application as a tumor inhibitor and may be used in vitro or in vivo to reduce the growth rate of neoplastic cells, particularly carcinomas and sarcomas of the lung, breast, skin, etc. A PF-4 formulation can be administered to a host suspected of having a neoplasm by applying the PF-4 formulation to the neoplastic site to reduce the growth rate. Application method: 1 local administration.

and systemic administration, including injection, introduction through catheters, etc.

Systemically administered formulations are generally known in the art and include formulation in buffered solutions or saline, or other suitable excipients. The dose is.

It varies depending on whether the administration is systemic or local. Dose concentrations are - at most about 0.1 ug to 1.0
00 μg/kg, and the total dose for large mammals, including humans, is approximately 0.0 μg/kg per treatment dose. O
It is 1 to 10 tg.

As will be apparent to those skilled in the art relevant to the present invention.

Variations of the above-described embodiments of the invention are intended to be included within the scope of the appended claims. For example, with appropriate replica and marker functions and appropriate restriction sites. ρB
I? It is also within the scope of the present invention to incorporate colicin E1 regulatory and structural gene sequences into plasmids other than 322.

(Left below) (Summary of the Invention) The present invention provides a method for improving the expression of a poorly expressed recombinant protein, such as platelet factor 4, by constructing a fusion gene. This gene fusion is &
This is done in such a way that undesirable secondary structures formed between the region of the mRNA that specifies the 11me protein and the region of the mRNA that contains the ribosome binding site are inhibited. Gene fusion involves inserting a segment of DNA of sufficient length to avoid unfavorable long-chain folding interactions (which keep the ribosome binding site inaccessible to the ribosome, thereby inhibiting mRNA translation). It is done to prevent The present invention further provides a recombinant DNA vector for expression of recombinant platelet factor 4 and a method for microbial production and recovery of the platelet factor 4.

Sat-Kokusara Summer■ Single-house escape Figure 1 C, CTAP-m mRNA O) First (9
The predicted secondary structure is 280 nucleotides. S-D is the ribosome binding site, and Ti is the translation initiation codon (AU
G) is shown. S-D and Ti sequences are shown in bold type.

Figure 2 is the predicted secondary structure of PF-4 mRNA.

The description of FIG. 1 also applies here.

Figure 3 shows the synthetic NA sequence synthesized for the construction of PF-4. The 12 oligonucleotides used to create the two main fragments (which upon ligation form the entire sequence encoding Pi4) are drawn onto the DNA sequence and
The joining site is indicated by an arrow.

A single change at nucleotide number 153 specifies asparagine or aspartic acid at this position.

FIG. 4 shows the plasmid pNP6-deltaEc used in preparing the expression vector containing the synthetic gene of FIG.
This is a restriction site and functional map of oRT.

FIG. 5 is a 5DS-polyacrylamide gel electrophoresis (PAGE) analysis of cell extracts showing the expression of several different colicin-PF-4 fusion proteins. lane l
is molecular weight standard; lanes 2 and 3 are 5 plasmid p
NP6: Col (504) - Plus and minus induction of PF-4: lanes 4 and 5 are plasmid pN
P6: Col (150) PF 4 ; lanes 6 and 7 are plasmid pNP6: Col (57
)-PF-4; lanes 8 and 9 are plasmid pNP
6: Col (5) Pi4i lanes IO and 11
indicates pNP6:PF-4.

that's all

Claims (1)

[Claims] 1. constructing a hybrid gene consisting of a first DNA sequence encoding the amino terminus thereof and separated by a cleavage site sequence and a second DNA sequence encoding a recombinant protein; A step of culturing a host cell transformed with a vector having the gene, and a step of recovering a fusion protein from the transformed cell,
A method for improving expression of a recombinant protein, comprising: as a first DNA sequence of the hybrid gene, an mRNA region encoding the recombinant protein and an mRNA region containing a ribosome binding site; DN of sufficient length to prevent formation of undesirable secondary structures that would make the site inaccessible.
A method comprising: selecting an A sequence. 2. The method of claim 1, wherein the DNA sequence selected as the first DNA sequence consists of between about 150 and about 450 base pairs. 3. The recombinant protein is platelet factor 4, the first DNA sequence is derived from the colicin E1 structural gene, and the carboxyl terminus of the colicin E1 structural gene is ¥E
cleaved at the co\R I restriction enzyme site, and \
Claim 1, wherein the Eco\RV restriction enzyme fragment is deleted.
The method described in section. 4. The method of claim 1, comprising expressing at least 10% of the total cell protein spacing protein. 5. Sequence that specifies the ribosome binding site and colicin E
The first gene encodes one structural gene and contains a translation initiation codon.
and a second R encoding platelet factor 4.
A composite mRNA having an NA sequence. 6. A recombinant plasmid expression vector for microbial production of platelet factor 4, comprising: a) colicin E having a promoter, an operator, a ribosome binding site, an initiation codon and a transcription terminator;
1 expression control sequence, b) a DNA fragment encoding a gene fusion consisting of a truncated colicin E1 structural gene, a specific cleavage site, and platelet factor 4, and c) an origin of replication. vector. 7. The plasmid expression vector according to claim 6, wherein the \Eco\RV restriction enzyme fragment of the colicin E1 structural gene has been removed. 8, pNP6-Col(150)-PF4 or pNP
The plasmid expression vector according to claim 6 or 7, which is 6-Col(57)-PF4. 9, ￥E. transformed with the plasmid expression vector of claim 8. coli￥. 10, Col(150)-PF4 or Col(57)
- A fusion gene product consisting of PF4. 11. A method for microbial production of platelet factor 4, comprising: a) ¥E of claim 9; b) disrupting the transformant; c) purifying the fusion protein from other cellular proteins; and d) culturing the fusion protein at a specific cleavage site. and e) recovering and purifying platelet factor 4 from the cleaved product of step (d). 12. The method of claim 11, wherein the platelet factor 4 is purified by heparin affinity chromatography. 13. Platelet factor 4 produced by the method of claim 11.